A number of infectious plant viruses, including members of the Tobamo-, Potex-, Poty- and Tobra-groups of viruses, share properties in common with one another. These properties include a single-stranded RNA genome encapsidated by viral coat protein oligomers that assemble to form either elongated rigid rods or flexuous threads.
Perhaps the best studied of the plant viruses is the tobamovirus Tobacco Mosaic Virus (TMV) which has a genome size of 6.4 Kb. The positive stranded genomic RNA codes for a number of viral proteins including those required for replication of the viral genome and those coding for structural proteins such as the coat protein which assembles into 20S protohelical or disk-like structures that become arranged into elongated helical structures with the viral genomic RNA molecule (Goelet et al., 1982, Proc. Natl. Acad. Sci. 79:5818-22).
Contiguous with the TMV genomic RNA is a sequence element referred to as the origin-of-assembly sequence (OAS) that is necessary and sufficient to direct efficient encapsidation of contiguous viral RNA sequences into virus particles. The TMV OAS is located approximately 1 Kb from the 3' end of the viral genome in the common strain (and in the coat protein gene itself in the cowpea strain (Cc; Sunn-hemp mosaic virus)) and consists of a 440 nucleotide sequence that is predicted to form three hairpin stem-loop structures (Turner and Butler, 1986, Nucl. Acids Res. 14:9229-42). The viral coat protein disks initially bind to loop 1 (the 3' most) during viral be assembly and in vitro packaging assays using mutant assembly origin transcripts have defined the 75 nucleotides comprising loop 1 as necessary and sufficient for encapsidation of foreign or viral RNA sequences (Turner et al., 1988, J. Mol. Biol. 203:531-47).
In vitro reconstitution studies have provided details on the assembly process for TMV. Preparations of purified coat protein, derived from virions from infected plant cells, are able to assemble into helical structures and virus-like rods, even in the absence of RNA at pH 5, suggesting that the coat protein contains the essential information required for self-assembly. Incubation of purified TMV coat protein preparations with TMV RNA at pH 7, in vitro, results in assembly of TMV-like viral particles containing encapsidated RNA (Fraenkel-Conrat and Williams, 1955, Proc. Natl. Acad. Sci. 41:690-98). Furthermore, it has been shown that foreign chimeric RNA molecules containing OAS sequences, transcribed in 30 vitro using SP6 or T7 (Jupin, I. et al., 1989, Nucl. Acids Res. 17:815) transcription plasmids can also be assembled in vitro into pseudovirus particles (Sleat et al., 1986, Virology 155:299-308).
Until recently, sources of viral coat proteins for in vitro reconstitution studies have relied on virus preparations made from infected plant tissue. However, such sources are disadvantageous since laborious procedures comprising infection with virus, purification of the virus from plant tissue, and then purification of coat protein from the virus must be used. The cloning and sequencing of a number of plant viral genomes has led to the identification of viral coat protein encoding sequences. Insertion of these genes into bacterial expression vectors has allowed the expression of, for instance, TMV coat protein in E. coli (Shire et al., 1990, Biochemistry 29:5119-26). However, it was reported (id.) that recombinant TMV coat protein produced in E. coli reconstitutes in vitro with TMV RNA at a greatly reduced rate relative to the reconstitution with native coat protein; the authors suggested that this inefficiency in reconstitution arises from the lack of an acetyl group on the amino terminus of the recombinant protein, which is present on the native coat protein. Zucchini yellow mosaic virus and Johnsongrass mosaic virus (both potyviruses, which are not members of the tobamovirus, tobravirus, or potexvirus groups) coat proteins have also been produced in E. coli (Gal-On et al., 1990, Gene 87:273-277; Jagadish et al. 1991, J. Gen. Virology 72:1543-1550).
Present work in molecular biology and recombinant nucleic acid technology is encumbered by problems associated with degradation of RNA by ribonucleases. Researchers in the past have relied on inhibitors such as human placental RNase inhibitor (RNAsin), or the use of alkylating reagents such as diethylpyrocarbonate (DEPC) which is a suspected carcinogen to inhibit the activity of ribonucleases; such inhibitors may produce (DEPC) undesirable modified components of RNA.